(i) Field of the Invention
The present invention relates to agents and methods for treating achondroplasia.
(ii) Description of the Related Art
Achondroplasia is one of the most common congenital diseases responsible for micromelic dwarfism characterized by short limbs relative to trunk. It is diagnosed by X-ray photographs in addition to growth failure in the major axes of the long bones of extremities and typical physical features such as a large frontally projecting cranium and a short nose. The disease is said to occur at an incidence of one to 10,000-25,000 people. This disease is an autosomal dominant hereditary disorder, but 80-90% of cases are found to be sporadic. Current therapies include orthopedic surgeries such as artificial hip joint replacement or leg lengthening and growth hormone therapy. Leg lengthening involves cutting bones at the age of 10 years or after and gradually increasing body height using a special device (leg lengthening device) over several courses of about six months. However, this procedure inflicts a great pain on patients. Growth hormone therapy increases body height by means of periodic growth hormone injections starting from childhood. However, growth ceases when injections are stopped. Neither therapy is curative, and neither are considered ideal from the viewpoint of patients"" QOL (American Journal of Medical Genetics 72: 71-76, 1997; European Journal of Endocrinology 138: 275-280, 1998). Consequently, it is desirable to develop a achondroplasia therapy based on a new mechanism.
Recent reports show that achondroplastic patients have mutations in fibroblast growth factor receptor 3 (FGFR3) located at chromosome 4p16.3, and two mutations are currently known. Of these mutations, 97% represents G1138A (change of 1138th G to A) and 2.5% represents G1138C (change of 1138th G to C), resulting in a change of the amino acid Gly at the 380-position to Arg (G380R) (Nature 371: 252-254, 1994; Cell 78: 335-342, 1994). To examine the relation of this mutation to achondroplasia, G380R FGFR3 (sometimes hereinafter referred to as FGFR3ach) transgenic mice were bred to provide an animal model for human achondroplasia. The mice showed short limbs and craniofacial hypoplasia (Development. 125: 4977-4988, 1998).
On the other hand, the natriuretic peptide (NP) family consists of three peptides, ANP (atrial natriuretic peptide), BNP (brain natriuretic peptide) and CNP (C-type natriuretic peptide), and is thought to show biological activity by increasing intracellular cGMP through two guanylyl cyclase coupled receptors (GC-A receptor for ANP and BNP, and GC-B receptor for CNP) (Annu. Rev. Biochem. 60: 229-255, 1991). NPs are reported to have important roles in the regulation of body fluid homeostasis and blood pressure control (J. Clin. Invest. 93: 1911-1921, 1987; J. Clin. Invest. 87: 1402-1412, 1994), but also they are known by their expression and physiological activity in various tissues other than cardiovascular system (Endocrinology. 129: 1104-1106, 1991; Annu. Rev. Biochem. 60: 553-575, 1991). Among them, they have a role as bone growth factor. In organ cultures of tibiae from fetal mice, CNP significantly promotes longitudinal bone growth (J. Biol. Chem. 273: 11695-11700, 1998). CNP is more potent than ANP and BNP in the production of cGMP in organ cultures of tibiae from fetal mice, cultured chondrocytes and cultured osteoblasts (J. Biol. Chem. 269: 10729-10733, 1994; Biochem. Biophys. Res. Commun. 223: 1-6, 1996; Biochem. Biophys. Res. Commun. 215: 1104-1110, 1995). CNP and its receptor GC-B are expressed in the growth plates of bones (J. Biol. Chem. 273: 11695-11700, 1998; Proc. Natl. Acad. Sci. U.S.A. 95: 2337-2342, 1998). CNP was also found to have a role in thickening the cartilage layer of the growth plate in transgenic mice expressing CNP specifically in cartilage (Yasoda et al., Abstracts of the 72nd meeting of the Japan Endocrinology Society, 1999).
The relation of CNP to dwarfism was also indicated because CNP knockout mice developed dwarfism (Proc. Natl. Acad. Sci. U.S.A. 98: 4016-4021, 2001), but nothing has been described about its relation to achondroplasia caused by FGFR3 mutations and no positive evidence has shown that CNP is effective for achondroplasia caused by FGFR3 mutations. That is, it is known that FGFR3 mutations are related with achondroplasia and that CNP is involved in chondrogenesis, but nothing has been known so far about the relation between them, particularly which of FGFR3 and CNP is located upstream in the regulatory pathway of endochondral ossification and whether or not CNP has a therapeutic effect for achondroplasia.
An object of the present invention is to provide novel agents and methods for treating achondroplasia caused by mutations in FGFR3.
On the hypothesis that a substance (e.g., CNP) activating guanylyl cyclase B (GC-B) may be applied to diseases involving chondrogenesis, we searched for a suitable achondroplasia model and mated this animal model with CNP-transgenic mice to prepare double transgenic mice for testing whether the symptoms of achondroplasia can be corrected. As described above, G380R FGFR3 (FGFR3ach) transgenic mice had been bred as an animal model of human achondroplasia, which showed short limbs and craniofacial hypoplasia (Development. 125: 4977-4988, 1998). Thus, we obtained such FGFR3ach-transgenic mice and mated them with our CNP-transgenic mice to prepare CNP/FGFR3ach-double transgenic mice, which were found to remedy the bone growth inhibition caused by FGFR3ach, whereby we achieved the present invention relating to agents and methods for treating achondroplasia with CNP.
Accordingly, the present invention provides therapeutic agents for achondroplasia caused by the cartilage growth inhibition resulting from mutations in the gene for fibroblast growth factor receptor 3 (FGFR3), containing a substance activating guanylyl cyclase B (GC-B) as an active ingredient, as well as methods for treating achondroplasia comprising administering a substance activating guanylyl cyclase B (GC-B).
As used herein, the expression xe2x80x9cachondroplasia caused by the cartilage growth inhibition resulting from mutations in the gene for fibroblast growth factor receptor 3 (FGFR3)xe2x80x9d means achondroplasia caused by hyperactivity or function control failure of FGFR3 or overexpression of the FGFR3 gene resulting from mutations in the FGFR3 gene, and achondroplasia is synonymous with chondrogenesis disorder. As used herein, FGFR3ach means fibroblast growth factor receptor 3 (FGFR3) containing a mutation of the amino acid Gly at the 380-position substituted to Arg (G380R), which is known to induce hyperactivity of FGFR3 (Development. 125: 4977-4988, 1998).
As used herein, the expression xe2x80x9csubstance activating guanylyl cyclase Bxe2x80x9d means a substance (peptide or low molecular compound) capable of binding to GC-B known as a receptor for CNP (C-type natriuretic peptide) to activate it, preferably a substance (peptide or low molecular compound) having CNP (C-type natriuretic peptide)-like activity, such as mammalian CNP (CNP-22 (Biochem. Biophys. Res. Commun. 168: 863-870, 1990, WO91/16342), CNP-53 (Biochem. Biophys. Res. Commun. 170: 973-979, 1990, JPA 1992-74198, JPA 1992-139199), avian CNP (JPA 1992-120094), amphibian CNP (JPA 1992-120095) and CNP analog peptides (JPA 1994-9688), preferably mammalian CNP, more preferably CNP-22. Identification of the xe2x80x9csubstance activating guanylyl cyclase Bxe2x80x9d is performed by, for example, expressing GC-B receptor in cultured cells such as COS-7, incubating the medium with a candidate substance (peptide or low molecular compound) at a given temperature for a given period (e.g., 37xc2x0 C., 5 min) and then determining the concentration of cGMP in the cell extracts (Science 252: 120-123, 1991).